The HIV -1 Tat protein is a transcriptional activator that is essential for viral replication. Identification of Tat inhibitors is likely to be facilitated by detailed understanding of the mechanism of Tat activity, its interactions with cellular and viral components, and atomic resolution structures of Tat and its relevant complexes. One key interaction is with the TAR RNA site found at the 5' end of the viral transcripts. The arginine-rich domain of Tat is primarily responsible for binding to a bulge region in TAR, with one arginine making an important sequence-specific contact to the RNA. An additional cellular protein, most likely cyclin T, recognizes the loop of TAR and establishes a high-affinity complex required for transcriptional activation. The Tat protein from a related virus, bovine immunodeficiency virus (BIV), recognizes a related TAR site but does not require an accessory protein for high-affinity RNA binding. Comparisons between the HIV-1 and BIV Tat-TAR interactions have been particularly instructive in elucidating the basic mechanisms of recognition and in suggesting possible strategies for designing tight HIV-1 TAR binding molecules. This proposal focuses largely on using structural information and combinatorial strategies to identify peptide and small molecule inhibitors of the HIV-1 Tat-TAR interaction. Specifically, genetic screens will be used to identify tight HIV-1 TAR RNA-binding peptides from combinatorial libraries, a dimeric BIV interaction will be designed to provide an additional starting point for developing tight HIV-1 binders, small molecules that potentially bind TAR will be identified using a computer algorithm that probes for specific hydrogen-bonding configurations in nucleic acid sites, and effects of potential inhibitors on Tat activation and virus expression will be measured in tissue culture assays. Additionally, the Tat-TAR system will be used to screen cDNA libraries for RNA-binding proteins. These studies may provide new approaches to inhibiting Tat function and HIV replication, and should enhance our understanding of Tat activation and RNA-protein recognition.